The lysyl-tRNA synthetase (LysRS) system of Escherichia coli consists of two differentially regulated genes. One gene, lysS, is constitutive, and the second, lysU, is inducible. The latter is a known cell stress gene, and its expression is induced upon heat shock. Of the E. coli aminoacyl-tRNA synthetases, LysRS followed by phenyalanyl-tRNA synthetase (PheRS) have been shown in vitro to be the most efficient in synthesizing adenylylated dinucleoside oligophosphates (ADO), a family of nucleotides of which the prototype is Ap4A. The intracellular concentration of the ADO rises markedly when E. coli, other bacteria, or eukaryotic cells are subjected to stresses such as heat shock, oxidizing agents, or heavy metal ions. It is important to determine if in E. coli the product of each LysRS gene, and/or PheRS are the predominant catalysts for the in vivo synthesis of the ADO. E. coli mutants defective for lysS or lysU or pheS as well as double lysS lysU mutants, and a triple lysS lysU pheS mutant are now available in this lab. This makes it feasible to examine if a single, double, or triple mutant strain will have a normal output of ADO, or if each LysRS gene product or PheRS might preferentially synthesize a subset of ADO. This work has the potential to establish a fundamental in vivo role for some aminoacyl-tRNA synthetases other than for the aminoacylation of tRNA. Additionally, if it is found that with each kind of cell stress a different tRNA synthetase is of particular importance in synthesizing ADOs or a subset of them, this would represent a type of metabolic code. The nucleotides will be labeled with 32-P-i prior to a stress, and samples will be taken at appropriate times upon application of the stress (heat, oxidation, heavy metal ions). The nucleotides will be resolved by two-dimensional thin layer chromatography using appropriate standards. Upon resolution of the nucleotides, the thin layer plate will be autoradiographed, and each spot which corresponds to an ADO will be cut out and quantitated by scintillation counting.